The mechanism through which copper is supplied to growing cells and tissues is at present uncertain and controversial. The present proposal will critically evaluate the hypothesis that ceruloplasmin is a major source of intracellular copper. Stated simply, the ceruloplasmin hypothesis postulates that (1) ceruloplasmin is a necessary if not obligatory source of copper for intracellular copper metalloenzymes such as lysyl oxidase, superoxide dismutase, and cytochrome oxidase; that the delivery of copper through ceruloplasmin is mediated by a surface receptor protein that specifically binds ceruloplasmin, and that uptake of copper is through a receptor-mediated mechanism that links the membrane with intracellular apoenzymes in the mitochondria and cytosol. Regulation of copper metabolism in part may be through control of the ceruloplasmin receptor. To evaluate this hypothesis we will use dietary copper deficiency to blunt the development of copper-dependent enzymes. The rapidly growing chick system will be ideal for such studies. The effects of copper deficiency on heart cytochrome oxidase will be studied in detail in hopes of determining the precise nature of the molecular impairment to the enzyme. That information that will impact on later studies that will investigate the mechanism through which ceruloplasmin restores functional activity to cytochrome oxidase. Other planned studies will attempt to isolate, purify and characterize the receptor for ceruloplasmin. Monoclonal antibodies prepared against the purified receptor will give insight into the prevalance of ceruloplasmin receptors in other tissues and factors that regulate their expression. Lymphocytes and cardiac cells will be grown in a defined culture medium supplemented with serum proteins. Such cells will be treated with antibodies to the ceruloplasmin receptor in hopes of blocking the ceruloplasmin-dependent uptake of copper. These studies permit a direct evaluation of ceruloplasmin on growing cells. They will also permit unequivocal evaluation of other copper complexes such as albumin-copper, Cu-GHL, and amino acids to restore function to the cytochrome oxidase complex in essence, to critically evaluate the necessity for ceruloplasmin in copper metabolism.